Copyright 0 1990 by the Genetics Society of America

Genetic Analysisof the Shaker Gene Complexof

A. Ferriis,* S. LLamazares,* J. L. de la Pornpa,* M. A. Tanouye? and0. Pongs* *Institute Cajal, CSIC, 28006 Madrid, Spain, +California Instituteof Technology, Pasadena, California 91 125, and 'Ruhr Universitat,Bochum, Federal Republic of Germany Manuscript received May 10, 1989 Accepted for publication March 3, 1990

ABSTRACT The Shaker complex (ShC) spans over 350 kb in the 16F region of the X . It can be dissected by means of aneuploids into three main sections: the maternal effect (ME), the viable (V) and the haplolethal (HL) regions. The mutational analysis of ShC shows a high density of antimorphic among 12 lethal complementation groupsin addition to 14 viable alleles. The complex is the structural locus of a family of potassium channels as well as a number of functions relevant to the biology of the nervous system. The constituents of ShC seem to be linked by functional relationships in view of the similarity of the phenotypes, antimorphic nature of their mutations and the behavior in transheterozygotes. We discuss the relationship between the genetic organization of ShC and the functional coupling of potassium currents with the other functions encodedin the complex.

HAKER was the first behavioral mutant detected do not appear tohave the capability of generating, by S in Drosophila melanogaster (CATSCH1944). It was themselves, gated ionic channels. named after another mutantwith a similar phenotype K+ currents are known to be themost diverse class isolated earlier in Drosophila funebris (LUERS 1936). of ionic currents in terms of kinetics, pharmacology The original phenotype was described as the trem- and sensitivity (HILLE 1984; RUDY 1988).Also, these bling of appendagesin the anesthetized fly. The elec- currents are known to play an essential role in many trophysiological studyof most viable Sh alleles has aspects of the biology of organismsfrom morpho- revealed a number of functional defects including: genesis (JAFFE 1979; KLINE,ROBINSON and NUCCI- excess of release at the neuromus- TELLI 1983) tomodulation of synaptic efficacy during cular junction (JAN,JAN and DENNIS1977), abnormal learning(LEVITAN 1988; KANDELand SCHWARTZ actionpotentials in the cervical giantfiber (CGF) 1982).In addition and in contrast toother ionic (TANOUYE,FERR~S and FUJITA 1981; TANOUYEand currents, K+ currentsare ubiquitous amongorga- FERR~S1985) and absence or altered kinetics of the nisms, tissues and developmental stages. fast voltage dependent transient K+ current (Ia) in Shaker was known to be a gene complex from the muscles under voltage clamp conditions(SALKOFF and beginning of its genetic analysis (TANOUYE,FERR~S WYMAN 1981; Wu and HAUGLAND 1985)well as as in and FUJITA198 1). We haveentertained thehypothesis single channel studies of dissociated nervous systems that thediversity of K+ currents has its counterpart in (SOLC,ZAGOTTA and ALDRICH1987). The subsequent the complexity of the Shaker locus. Now that one of molecular analysis of this locus has shown the exist- the originalpropositions has beendemonstrated, ence of a complex transcription unit from which a namely that ShC is the structural locus for a number large family ofproducts is generated by meansof of K+ channels, it seems appropriate to dissect ShC differential and/oralternative splicing mechanisms into its genetic components, to study the biology of (BAUMANNet al. 1987; TEMPELet al. 1987; KAMB, their mutations and tobegin to unravel the functional IVERSON andTANOUYE 1987). Most of these products relationships among thesecomponents. Thereare have structural features compatible with membrane abundant examples in a variety of preparations illus- proteins (PONGSet al. 1988; SCHWARTZet al. 1988) trating how K+ currents are modulated by means of and some of them have beendemonstrated tofunction phosphorylation,neuropeptides, cyclic nucleotides, as K+ channels afterRNA expression in Xenopus etc. (LOGOTHETISet al. 1987; NORTH et al. 1987; oocytes (TIMPEet al. 1988; IVERSONet al. 1988). ASHCROFT1988). Synaptic efficacy is largely based on Different RNAs give rise to K+ currents of different the diversified modulationof K+ channels (CROW characteristics depending on the specific 3' and 5' 1988; LEVITAN1988). In this context wewill ask if exon combination present (KAMB, TSENG-CRANKand the requiredfunctional coupling among these diverse TANOUYE1988). Thus, it seems that this transcription activities is related to thegenetic organization ofShC. unit from Shaker encodes a variety of K+ channels as We do not define Shaker solely on the basis of the well as a number of putative membrane proteins that K+ currents phenotype. Also, we do not assume that

Genetics 125: 383-398 (June, 1990) 384 Ferriis et al.

the Sh products participate exclusively in the forma- tizedflies were observed for 30 min or longer during tion of ion channels. We find untenable such a one- recovery under the dissectin microsco e The following Sh to-onecorrespondence between geneproducts and viable alleleswere tested: ShF , Sh'", Sh"';' and ShrK"'*O. Electrophysiology: Action potentials were registered biological features. Rather, we define a genecomplex from the CGF interneuron (KOTO et al. 1981). The proce- in broader terms as the portion of the DNA where dure for dissection, experimental conditions and character- clustered mutations show genetic relationships andl isticsof the preparation canbe found in TANOUYEand or similar phenotypes at any given level of observa- FERR~S(1 985). tion. Mosaics: Gynandromorphs were obtained from the prog- eny ofcand/cand females or among R(I)Z,ln(I)w""/*embryos. The resulting mosaics were routinely analyzed for general MATERIALS AND METHODS behavior including possible shaking activity. Also,the extent of maleffemale territories were drawn on standard fly Mutants, rearrangements, mutagenesis and nomencla- sketches. Each gynandromorph was either mounted in Eu- ture: The description of mutants and rearrangements used para1 for detailed cuticular observation or processed for in this study can be found in LINDSLEYand GRELL(1968), histology. A tentative fate map of the mutant focus of each LINDSLEYand ZIMM (1985, 1986, 1987) or TANOUYE,FER- lethal was calculated with the limited collection of gynan- Rirs and FUJITA(1981). Also, Table 1 and Figure 1 show a dromorphs as described in HOTTAand BENZER(1 972). summary of the rearrangements used. Somatic recombination cloneswere induced by X-ray The following agents were used as mutagens under the (Philips MG 151 Be, 150 r/min, 100 kV, 15 mA and 2 mm conditions of LEWISand BACHER(1 968) or AUERBACHand AI filter at a total dose of 1000 r) in heterozygous larvae of MOSER (1953): ethyl methanesulphonate (EMS) (250 mM), the indicated age. Germ line clones were induced by the ethyl nitrosourea (ENU) (250 mM), formaldehyde (F), die- same procedure in FS(I)KS1237 v/* larvae irradiated 24- poxy butane (DEB) (50 mM) and X-ray (4000 r). Unless 48 h after egg laying. The use of this agametic dominant otherwise indicated, 4-7 days oldf' os males were treated. female sterile mutant (FS(I)KS1237)allows the detection of The T(X;Y)'s wereinduced by J. Merriam on ay+P.YLBs a germ line clone bearing female as a fly that under COP chromosome. The proximal and distal elements canbe anesthesia relaxes the genital muscles and liberates a re- obtained separatedly and are designated by superscript P or tained egg. Under these conditions 6% of the irradiated D. Males of the constitution X/V7'/Y are sterile due to the control females carry a germ line clone (WIESCHAUS1980). hiperploidy of the base of the X. We mutagenized YsX.YL The development of each egg laid by these mosaic females compounds with X-ray in order to obtain deficiencies of the was analyzed individually. base of the X chromosome as fertile males YsX YL*/V7'. In Lethal phase and whole mounts: Fecund females were this way Dj(I)S4010was obtained. In(I)Px (Panoramix)was allowed to lay eggs for 20-hr periods at 25" and 80% obtained from the progeny of X-ray treated os males and humidity on regular fly food plates containing 5% sucrose detected by thedominant, larger than normal, eyesize and a drop of live yeast. Eggs were collected, counted and phenotype. transferred to petri dishes containing filter paper soaked in The mutations located within ShC are named after the 5% sucrose and a few drops of yeast. Groups of 20-30 eggs region to which they map: ME, V or HL for maternal effect, were examined at 1-day intervals; thus, the fraction of dead viable and haplo lethal respectively, followedby the number individuals was estimated in 24-hr periods. Routinely, the of complementation group and the code number. For in- lethal embryos were cleared and mounted for observation stance, ShMI.".3u5indicates a Shaker of the first group (VAN DER MEER, 1977). The classificationof lethals as of complementation (from distal to proximal) in the ME recessive, semidominant or dominant is according to region, isolated as code number 305. HADORN(1 955). Characterization of the shaking activity. The intensity of appendage vibration in the various genotypes listed in RESULTS Table5 was rated1, 2 or 3 in increasing order.These estimations are based in the shaking activity of the meso- thoracic legs of groups of ether-anesthetized (30 sec) flies. Aneuploid analysis:In the process of analyzing the Care was taken to use flies not older than five days and not ShCwe have used a number of chromosomalre- younger than one day. Aged Sh mutant adults (over 10 days) arrangements (see Figure 1) with breakpoints in the exhibit progressively more intense, although erratic, appen- vicinity of the Shaker locus. In order to identify their dage vibration, possibly related to the conspicuous impair- linear order and the existence of lack-of-function le- ment of muscle structure observed in several Sh alleles. Also, very young Sh adults (1 day) do not express the shaking thals, we constructed aneuploids in all feasible com- phenotype in full magnitude. The rating of the shaking binations. A summary of the information obtained is activity of any given genotype was decided after the obser- shown in Figure 1. vation of a minimum of 20 flies from different crosses and To dissect the 16EF region by means of aneuploids observed during the entire process of recovery from ether we constructed males Df(l)JC153/W32' and anesthesia. Whenever possible, control sib adults were co- anesthetized with the mutant genotypes. Df(l)JC153/B55p. These males are viable (10-20%) Assayswith other anesthetics (N2, Cl+C, COS and cold with respect theirto sib males FM7cIW3Y; temperature) were carried out under the same conditions Dp(l;3)JCl53/+. We chose the latter as control be- and yielded similar results as ethyl ether. However, anes- cause theycorrespond tothe homologousgamete thesia with triethylamine ("FlyNap" Caroline Biological segregation in the females of the cross: 0 Supply) yielded results that differ among the Sh mutants (see results). The procedure of anesthesia withtriethylamine T(X;3)JCl53/FM7c/TM6 X 8 W32/0. A similar crite- was as follows: an impregnated brush was introduced in a rion was followed in the cross: 0 Df(l;3)JCl53/FM7~/ 100-ml vial with 20 flies for 5, 3 or 1 min and the anesthe- W32'; Dp(l;3)JC15?, Ki SblTM3 X 8 CS. In practice, GE NE S REARRANGEMENTS NOTES BANDS GENES REARRANGEHENTS NOTESREARRANGEHENTS GENES BANDS BANDSNOTESREARRANGEMENTS GENES

m(1)px 05 17A 1 11 Df(1los IA 'A' deletes 16A B Tf1;4JB Dfillos '9130 &"-- - ' +17516 31 4

z G Dp(1:l)Bx r Distal break at 1503 4- Dfi1JB25 i5 DP(1;IJBx r49k !F t 190 kb Df (1)B 263-20 Distal break at 15F1-2 B I"-- Df'11N19 9} -17516 - Df(1)os 'A (1) (2) TIX:YJB75 B&z!FTL TfX;YJV29TfX:Y)Bl37 (3) TfX:YJBIB 2 TfX:YJB13Z 3 4 \ TiX;3)JCI53 1 This breakpoint is lethal 5 c111 6 ,i 1584 1- 1- Dp(1:31A59 Distal break at ';I BX T1X;YJBlO '3kff Dp(I:l )Ex r49k if"--- q-" 7 7 8 Dl 9 2 16 3 f" Dl 4 2 5 3 4 5 (4) 6 7

DP(1;ljBx r E :+" T(X;3)JC153 TfX:Z)BZ7 100 kb ME

FieTiX:3JSh Lc E~~F1 31 1 TiX;YJB55 I 70 kb V y- -TiX:YJW3Z MP hdp is covered by W32 p -1n(1)9916 : 3" 175 kb HL w TiX:YJv7 t1

BANDS GENES REARRANGEMENTS NOTES

Dfiliy 4L sc 9R (5) ''A: ''A: Df f 1 J JA27 Proximal break at 18D1-2 5 eTfX;YJRI (6) 6 7 Df (1jN19 81 2 3 4 5

i LDfflJy 4L SC 9R 10 11 c1 2 3 4 5 6 7 8 Di 1- Df(lJJAZ7

1 T(X;Y)R34 8 ]= 9 10 11 H(l)n 12 13 El 2 3 4

DplXrY)mal IO6 ';I- ';I- 5 FIGURE 1.--Aneuploid analysis of the 16A-19A region. The entire DNA covered by the Dp(1;3)JC153has been cloned and theencompassed breakpoints have been located in the corresponding restriction fragments. The sterility of hyperploid males X/xP (from the T(x;Y)'~) is caused by the duplication of the 18D region. * = Predicted lack-of-function lethal. (1) S137p/Df(1)N19males are letha]. (2) The v29 breakpoint is located 20 kb distal to B137. (3) B137D/V29P or E137p/V2pmales are viable while B137D/B132!' are letha]. (4) Tu/Dp(l,.l)Bx' and TU/DP(~;~)BX"~~females are phenotypically Tuf. (5) RIP does not cover Df(1)y'" sP. (6) RlD/Df(l)N19females and ~1~/~f(1)~,427 males are lethal. 385 386 FerrGs et al.

A B ploid is the deficiency while the second is the corre- 12 12 sponding duplication. The cloning of the DNA en- compassing these breakpoints has proven this point (BAUMANNet al. 1987). The aneuploid W32"/V7' is lethal in males as well as in heterozygous females (Figure 3). The dominant lethality of this deletion can be rescued by one dose of the Dp(1;3)JC153 or the fragment W32'. We name this interval haplo lethal region (HL). This is the only HL region that we have detected between 15D and the centromeresince females V7"/+, Df(1 )B25/+ and B75"/T(X;3)JCl53/W32' (see Figure 1) are viable. For the purpose of the analysis of ShC it is important tonote that the interval V7-JCI 53 proximal (14 bands) includes a minimum of three lack-of-function lethals since the aneuploid males Df( l)N19/B137', Bl37"/Bl3Y and T(X;3)JC153(see Figure 1) are not FIGURE2.-ME- males are viable. Southern blot of Hind111 viable unless the additional fragment V7' is present. digested DNA fronl CS (lanes 1) and Df(l)JC153,v/W32";TMI/+ Also, it should be noted that the hyperploid males (lanes 2) males hybridized with probes from the ME region (panel A) and from outside the ShC (panel B). The probe used in panel A X/Dp(l;3)JC153/Dp(l;3)JC153/B18'are viable and is the cDNA adm 135H8 from coordinates -1 ..5 to 3.5. The probe do not show a shaking phenotypeunder visual inspec- used in panel B is lambda Bb.5 clone from coordinates 3 13 to 33 1. tion nor in the CGF action potentials (data notshown). The experimental males are deficient for the interval -76/-73 to Extent and constituents of the ShC Among the 98/99.5 in the DNA map (Figure 3). Each lane contains the DNA available rearrangements with breakpoints in the re- extracted from 21 single male fly and both hybridizations were performed in the smle blot. gion (Tablel), T(X;Y)B55,T(X;3)ShLC, T(X;Y)W32 and T(X;Y)V7 show a noticeable shaking phenotype. the experimental as well as the control genotypes are Studies of K+ currents on muscles under voltage clamp scarce probably because other gametesegregations conditions have shown that W32 eliminates Ia, while are favored. Molecular evidence for this deficiency B55 manifest a residual current andV7 does not affect was obtained in Southern blots of Df(l)JC153,u/W32' it (SALKOFFand WYMAN1983). By contrast, B55 is a males probed with a DNA fragment from this interval more vigorous shakerthan W32 and V7 is clearly (Figure 2). However, the viability of these deficient distinguishable from the normal type (see Table 5 males is dependenton the maternal genotype. At- below). Also, the action potentials fromthe CGF tempts generateto these males from females interneuron are abnormal in the four cases (Figure 4) Df(l)JC153/FM7c/W32' or Df(l)JC153/FM7c/B55' (see also TANOUYE,FERR~S and FUJITA 1981). In have failed. In these cases the maternal genotype was agreement with the shakingactivity, B55 shows a more monosomic for the interval in question while in the defective than W32 (Figure 4). The crosses described above, the maternal genotype was discrepancies between the phenotypedefinition at euploid. Based on these observations, we named the these three levels of observation probably reflects the interval JC153-W32 (Figure 3) as maternal effect re- diversity of biological functions encodedin the region. gion (ME), indicating that the nullosomy for this re- The nearest breakpoints withouta shaking phenotype gion is tolerated only if the normal (disomic) amount are those of JC153 at thedistal end andS4010 at the of products were supplied during the oogenesis. A proximal terminus (Figure 3). Thus, it can be said further dissection of this region allowed to restrict the that theShC is included within the interval defined by ME region to the interval JC153-B55. The viability the latter two rearrangements. of ME- males implies the absence of lack-of-function The Dp(l;3)JCl53 covers about 22 bands. In order lethal mutations in this interval (however, see below). to identify the geneticcomponents of the region Adjacent and proximal to the ME region we define where the ShC resides, we set out a lethal saturation the viable region (V) on the basis of the viability of experiment. Mutants were detected in a F2 screen the aneuploid males B55"/W32' as well as B55'/W32D and isolated over the Dp(1;3)JC153 according to the (Figure 3). These males are viable irrespective of the procedure of Figure5. Thisprocedure allows the maternal ploidy for this region. Asin the previous isolation of all locimutable to lethality in this interval. case, the viability of both aneuploids implies the ab- The aneuploid analysis (see above) had indicated the sence of lack-of-function lethal mutations in this inter- existence of at least three lack of function lethals in val. The stronger phenotype of B55"/W32' versus the interval V7-JC153 proximal. Also, adominant B55'/W32" (Figure 4) indicates that the first aneu- lethal would be expected in the HL region. Finally, Genetic Analysis of Analysis Genetic Shaker 387

LC JC153 n27 US5 Sh W32 V7 SI010 Px N19 V29 b137 X153 I I 1 d I Shaker gene complex "-

ME v HL

FIGURE3.-Extent of the Shaker gene complex. The rearrangements used (see also Table 1 and Figure 10) are positioned with respect to their location in the map of the cloned DNA (upper scale). Those marked by arrowheads were used to dissect the region into three main zones: ME, V and HL. The proximal limit of ShC resides somewhere between V7 and S4010.

ME, V and HLregions appear to house a high density of functions with respect to the remaining DNA cov- ered by Dp(l;3)JC153. 9 The corresponding allelomorphism test among the B55 W32 i v7 isolated mutations revealed three complementation groups namedMEI, I1 and I11 from distal to proximal, as components ofthe ME region(Figure 6). The 140mV finding of lethals in the ME region is an unexpected "\ -I: \--4ms result in view of the viability of ME- males. In fact, B55D/W32p ~55'/~32~ this is the most frequently mutated region. None of the isolated lethals is rescued by extra doses of the FIGURE4.-Action potentials from rearrangements andaneu- ploids within ShC. Recordingswere obtained from the CGF of ShC+ in the maternal genotype. Indeed, the proce- males after brain stimulation. V7 shows two traces, one of them dure of mutant isolation precludes the detection of below threshold. Df(I)B55D/W32pshows three traces, one ofthem mutations rescuable by maternal hyperploidy (Figure below threshold. Note that Df(Z)B55D/W3Yhas a longer repolari- 5). On the other hand, since some of the alleles have zation delay than the Df(I)B55p/W32Dsuggesting that a product(s) been induced by X-ray, it is reasonable to assume that is encoded in this interval and its lack-of-function contributes to the abnormal profile of action potentials. Also, note that Dp(1)B5ZP/ some of them are bona fide lack-of-function lethals W3ZD shows a more severe phenotypethan W32 suggesting that the (see DISCUSSION). It is evident that thelethality of these aneuploid perturbs the function of more products than it does W32. mutations is not a phenotype equivalentto their dele- the ME regionshould not contain lack-of-function tion. It can be concluded that the lethality of the ME lethals because of the viability of ME- males. mutations is an antimorphic trait(MULLER 1932) (see A total of 19902 fertilechromosomes were also below). screened in the F2 generation after treatment with As expected, in the V region no lethals were found. various mutagens (Table 2) and 33 lethals were iso- Inthe HL region, the mutagenesis with alkylating lated. These lethals were mapped with respect to the agents, that usually producepoint mutations, has available breakpoints by constructing the aneuploids failed to generate theexpected dominant lethals. Only 8 */W32p; 8 */B55'; 8 */V7'; 0 9 */Df(l)N19 when X-ray was used, 3 dominant (DL) and 1 semi- and c3 */V2f. The abundance of lethals within the dominant (SDL) lethals were found (Table2). It seems ShC vs. the interval V7-JC 153 proximal is a remarka- that a major disruption of the genetic organization in ble feature (Figure 6). It seems that the lethal muta- HL is required to obtain dominantlethals. Also, while tional target of the JCl53 distaLV7 region is much the ME region has three complementation groups, the larger than therest of DNA covered by Dp(l;3)JC153. HL region contains nine. However, both regionsspan Since the entireDp(l;3)JCl53 has been clonedwe can over a similar length of DNA (Figures 3 and 6). The say that the 350 kb of ShC have yielded 29 lethals DL mutations can not be tested for allelomorphism while the 190 kb outsideof it (Figure 6) have yielded because of their dominant lethality. It is not known if only 4 lethals in the saturation experiment. Thus, the the three DL mutations correspond to thesame com- 388 Fer& et al.

TABLE 1 Rearrangements in the region of ShC

C hromosome Breakpoints Markers Breakpoints Chromosome Notes References" T(X Y )B75 16B/Y" xp,y+; X",y 3 Viable.a Needs Y chromosome V T(X; 3)JC 153 16E/17A12-B1 u breakpointProximal is lethal + T(X 2)B27 16E4-F2/36D-F B ShK"j3 infrequentViable although 4 T(X Y)B55 16F1-4/Y" X",yBsXp,yy+; a Needs Viable. Y chromosome V T(X Y)W32 16F3-6/Y" Xp,y'; XD,yfertile wf' B" Viable and V T(X; 3)ShL' 16F1-2/80 dnc2 4 T(X Y)V7 16F5-8/Y" XP,y+;XD,y wf' B" fertileViable and V Df(I)S4010 17A/20 YWUf' on Induced a Y'X.Y L compound. Lost 4 In(1)Px-2/8C 1 17A os Punorurnix (Px) causes abnormally large eyes 4 Df( 1)NI9 17A7-8/18A6-7 + T(X;Y )V29 17A10-11/Y1. Xp,Bs;X",y wf'y+ V T(XY)B137X",y 17A10-ll/Y" Xp,y'; B' V T(X,Y)B132 17All-I2/Y" xp,y+;X",y B V v =J. MERRIAM(personal communication); 4 = J. LEFEVRE(personal communication; 4 = this work.

5 P 88 f os (~tageaixed)X 99 c(lJn3; DP/~U TABLE 2 Lethal saturation mutagenesis

5 I1 1:1 -d f 0. f Dp/+ "Mutagen Lethals numberCode X 2,855 13bEMS 162, 174,581,387, 305, 583, 598, 1199, 1359, 93 C(1)10 f Dp/ln3 157V, 1614, 1929, 2215 1,166 ENU 5 484,2288,2496,459, 5 if Po -Ud f 0. : nu/+ 301 4 5 502 DEB 0 take Ud f w*:Dp/ln3 1,462 F 0 X 13,917 X-ray 14 254,2270,4058,5051, 99 CflJN3 I Dp/XW 7688,8384, 9130, 9916, 12748, 13167, 13193, 15175, 17053, -STOCK I7516 FIGURE5.-Lethal saturation procedure. TheDp(1;3)JCI53 (ab- Spontaneous 1 17266' breviated Dp) covers theentire ShC andthree lack-of-function 19 ,902 33 (29 3319,902 of them within ShC) lethals proximal to ShC and os (see text and Figure 1). The muta- genized chromosomes are markedby *. Occasionally os males were The number of sterile chromosomes (averageof 2 1%) has been used instead off' os, but both chromosomes areof the same origin. deducted. Since the maternal genotype is hyperploid for ShC+, no maternally 'The frequency of mutability of ShC (about 4.5 X lo-") can be rescuable lethals could be detected. The single male crosses of the compared with that ofyellow = 1.7 X lo-'' or with that of rosy = 1.5 x IO+. F1 generation were screenedfor the absence off os; TM3/+ ' 17266 is a gift of A. SCHALET. indicating the presence of a lethal covered by Dp(1;3)JC153. Also, The mutations 5051,9916 and 13193 are dominantlethals; 8384 these crosses were inspected for visible mutants. is semidominant and the rest arerecessive (however see also Figure 7). EMS, ethylmethanesulfonate; ENU, ethylnitrosourea;DEB, die- plementation unit nor if they are allelic to any of the poxybutane; F, formaldehyde. other recessive lethals. Thus, the actual number of known. The case of 174 and 1614 is particularly HL complementation groups could be higher. interesting. These two mutations belong to different Recombinationalmapping among selected repre- complementationgroups and can beseparated by sentatives of the complementation groups was under- recombination. The exchange of flanking markers in taken (Figure 6). In general, the values of the fre- the recombinants unequivocally locates I74 distal to quencies of recombination are in agreement with the 1614 (see legend of Figure 6). However 174 fails to span of breakpoints and theDNA content known after complement 4058 which is locatedadjacent to V7 cloning the area (Figure 3), roughly 1 cM = 1 Mb. accordingto Southern blot analysis (I. KRAH-JENT- The linear order of mutations in the ME and V GENS et al., submitted for publication). Because ele- regions was ascertained because the flanking markers ments atboth extremes of theHL region fail to in the heterozygouscombination of mutantswere complement, the whole region must be considered as different (see legend Figure 6). However, the linear a functional unit. order of most lethals within the HL region is not As atest fordetecting possible rearrangements " - 0.01 0.01 0.01 0.1 0.1

0.03 0.04 0.05 - 0.09

0.2

0.1

0.2

n. 2 FIGURE6.-Mutant saturation of the Sh region. Lethals are marked by a code number and the mutagen of origin by the symbols: A = EMS; A = ENU; 0 = spontaneous or = X-ray. The linear order of ME lethals is based on the exchange of flanking markers in the recombinants obtained. The most frequent combination of flanking markers used was: y w f ""*fly* os. The linear order of the HL lethals (box) has not been determined exceptfor 174/1614. The dominant lethals (DL) 5051, 9916 and 13193 can not be adscribed to a complementation group because of this feature and show 0 viability in heterozygous females. The semidominant lethal (SDL) 8384 is poorly viable (about 30%)in heterozygous females and show 0 viability over 1614. Also, it shows a wings up phenotype over hdp. The recombination frequencies are based on a minimum of two recombinants after pooling data from several crosses. It should be pointed out that this is an "attempt"of mutant saturation. It is not possible, at this time, to know if all existing complementation groups have been identified nor if all existing transcriptional units have been mutated. among the isolated lethals we screened the salivary vae f * os/y w irradiated at 48-72 or 24-48 h before gland chromosomes (2215, 459, 2288, 3014, 838dSDL, puparium formation. No mutant spots were recovered 9Y16DL,5051DL, and 131Y3DL)or measured the fre- while 11 notum, 9 eye and 34 abdomen control twin quencies of recombination between flanking markers y w clones were found, indicating that 305 and 2215 (305, 13359, 387,1929, 174, 484, 1614, 2288, and are cell lethal mutations (RIPOLL1977; BRYANTand 5O5lDL).It was found that 9916DLis an inversion with ZORNETZER 1973.). Similarly, the attempts to generate breaks at 16F and 14A.2288 seems to be a very small germ line mosaics (see Material and Methods) have deletion of the proximal part of 16F. This mutation failed (Table 4). The cell lethal condition of these reduces the frequency of recombination sixfold be- mutations precludes anyfurther characterization. tweenffand ShKS'33.Also, 5051DLreduces by 30% the The ME11 group is defined by the mutations 162 recombination in the interval f-os suggesting that, and 1359. The latter was studied in detail. The major although not detected by us in the polytene chromo- component of the letal phase occurs at the late pupal somes, this mutation might be a subtle rearrangement. period(Figure 7), however noadult escapers have The rest of mutations tested do not alter the chro- ever been detected. The lethality appears to have a mosome banding nor the recombination frequency. slight dominant componentsince the fraction of lethal Biology of ShC mutants: Characterization of the individuals in an outcross is higher than the expected isolated mutations consisted in the study of the follow- 25% (Figure 7). The mutant territory in gynandro- ing traits: CGF action potentials, lethality phase and morphs shows defective bristle and vein patterns., mosaic analysis. The types of mosaics studied include Also, the mutant region of these mosaics exhibits a gynandromorph, somatic and germ line clones. noticeable shaking activity (Table 3). Finally, the germ The ME mutants: The ME1 group is defined by the line clones are obtained in anormal frequency al- mutations 305,254,1579 and 2215. The study of 305 though the resulting embryos fail to develop irrespec- and 2215 shows that, although both mutations belong tive of the zygotic genotype (Table 4). These abortive tothe samecomplementation group,their lethal embryos do not progress beyond 40% embryogenesis phases are different (Figure 7) suggesting that several in contrast with the relatively late lethal phase ob- functions might be encoded in this complementation served in regular outcrosses (Figure 7) (see DISCUS- group. Also, 2215 complements only partially the SION). Since 50% of these embryos should be 1359/+ adjacent mutation 1359. The corresponding hetero- females, it can be concluded that the oogenesis from zygous females show a Minute-like phenotype. The a 1?59/1359 oogonia is defectivefor an essential ME1 mutations are lethal in gynandromorphs (Table maternal function which can not be supplied by the 3). We attemptedto generate smaller mosaicsby normal genome of the zygote. means of somatic recombination in heterozygous lar- The MEIII group is defined by eightmutations 390 Ferrbs et al.

305 22 15 (Figure 6). We have studied 387, 583, 598, 1929 and I I 459 as representatives of thegroup. Their lethal phases are very similar in all cases (Figure 7) with the possible exception of 459. The latter exhibits a clear 25 dominant shaking activity in heterozygotes. However the inspection of polytene chromosomes,measure- n- I30 6-322 n-99 6.31% ment of recombination frequency between flanking 1359 38 7 markers and Southern blot analysis has yielded no 'O0- 'O0- 75 evidence for a rearrangement that could affect simul- taneously the ME and V regions. Similarly, 387 ex- hibits a mild to weak shaking activity in heterozygous 25 25 females. In this case, we registered the CGF action O E I II 111 LPEP E I II 111 EP LP potentials and found them to be abnormal (Figure8) n- 109 6.42% 11.123 6.362 resembling some viable Sh alleles (see below). For 583 598 ." the gynandromorph analysis we used 1929. The 16 lo01 mosaics obtained show normal hypodermal differen- I tiation.However, 12 of themhad at least one leg 25 ;:L25 abnormally positioned or with shaking activity (Table 3). The germ line mosaics (Table 4) from the five O E I II 111 EP LP O E I II 111 LPEP n-706-29U alleles tested indicate that the mutated MEIII func- 1929 tion(s) do not alter oogenesis. An exceptional case, however, is 1929. In this case the homozygous ova- rioles yield viable mutant males (Table 4). Under the usual maternal genotypes (*/FM6) as well as in out- 25 crosses (*/+) these mutations show complete lethality.

E I II 111 EP LP It appears that, when oogenesis is carried out by cells n-896.342 n-137 d.242 containing a mixtureof mutated and normal products, which is the case of heterozygous mothers 1929/+, 2288 484 the maternal effect on the development of male 1929 zygotes is more deleterious thanwhen the cells contain loo- 75 only mutated products. This phenomenon is indica- 751 I looi tive of the antimorphic nature of the mutation and 25 also a suggestion of the involvement of MEIII func-

O E I II 111 LPEP tion(s) in oogenesis. Furthermore, it calls for a mech- n-101 1.31% n-92 1.13% anism by which the normal MEIII products give rise 1614 8384 to multimeric structures (see DISCUSSION). loowFinally, the ME region harbours also the site of the 75 viable rearrangement T(X;2)B27(Figure 3). This mu- tation was induced in a ShKS133chromosome and de- tected as a modifier of the shaking phenotype. In the :"II.L25O E I II 111 EP LP doublemutant, the shaking activity is of ahigher n-82d-22% 11.458 6-442 frequency and amplitude than in ShKs133.Since the 174 4058 breakpoint of this translocation in chromosome is 100, 1 00 I-. ZZ in thecentromeric heterochromatin (Table l), the dominant enhancing effect is most likely due to the functional modification of the X chromosome com- 25 250 E I II 111 EP LP ponent. The subsequent study of the CGF action O E I II Ill LPEP "d_l potentials of this doublemutant show the typical n-77 1.35% n-150 6-262 30 14 505 1 FIGURE 7.-Lethal phase of ShC mutants. A total of tt eggs from 100 T"------l outcrosses 0 */+ X 8 + was followed (see MATERIAL AND METHODS) during development and the total percentage of dead individuals (d) was distributed in the corresponding instars. Mutants in the upper half of the figure belong to the ME region and those in the lower half belong to the HL region. Note that some lethals (1359, 387, 8384 and 5051) exhibit significant dominance in this trait. E n-BO 6.312 n.267 6-382 = embryo, LI-111 = larval stages, EP = early pupae, LP = late pupae. Genetic Analysisof Shaker 39 1 TABLE 3 Gynandromorph analysis

~ ~ ~~ ~~ Region phenotypeMutantHypodermal activity n Shaking Control ME 305 0 46 2215 0 32 1359 18 52 ++ Bristles short, disoriented and fewer; plexated veins 1929 16 61 ++ Normal; abnormal position of wings and legs HL 1614 14 23 ++ Normal; abnormal position of wings and legs 174 18 174 Very few and short bristles. Gynandromorphssmall with male patches 2288 13 43 + Bristles short, disoriented and fewer; plexated wings 3014 23 35 - Bristles short; sluggish V shKSI33 70 64 +++ Normal Mosaics were obtained from the crosses: 0 ShKSi3'/FM6;cand/cand X 6 y w and 0 f"os /FM6 X d R(1)2,In(1)w"". n = experimental mosaics. Control = 0FM6/y w or 0 FM6/R(1)2,In(l)w"',' mosaics. Shaking activity refers to any appendage. The fate map of this behavior (HOTTAand BENZER1972) indicates independent foci for each leg and a ventral location (data not shown).

TABLE 4 Germ line mosaic analysis

~~~~~~~~~~~ ~~ ~ Sh region Lethal n Clones Notes ME1 305 376 2" Germ line lethal 174 2215 319 0 Germ line lethal 1614 ME11 1359 259 21 Eggs do not develop inde- pendently of the zygotic genotype MEIII 387 130 Zygotic lethal 583 38 Zygotic lethal 598 210 Zygotic lethal 1929 369 1 1 clones yield 10% 1929 387 adults FIGURE8.-Abnormal action potentials from ShC lethals. Re- 459 90 Zygotic lethal cordings were obtained from the CGF off' 1614 0s/B137~;~wf3"' 174/FM6 andf" 387 os/FM6 females. Similar results were obtained HL 174 208 Germ line lethal inf" 1614 oslFM6. Since the chromosomal background of origin 1614 344 10 clones yield 14% 1614 and the X homolog are different in these genotypes, the possibility adults that an unknown dominant factor be the cause of the abnormal 484 182 7 clones yield 25% 484 action potentials appears very unlikely. adults 3014 207 11 Zygotic lethal 2288 21 7 1" Germ line lethal breakpoints of B55 and W32. Based on gene dosage experiments we had located some viable Sh alleles in a Recombinant took place distal to the lethal locus as proven by the analysis of the offspring. Viabilityof the lethal escapers is this interval(TANOUYE, FERR~JS andFUJITA 1981). calculated with respect to the sib females FM6/*.The escapers lived Since the mutations in this region are viable, the for a few days with progressive motor impairments and failed to behavioral and electrophysiological analyses have mate. n = number of irradiated FS(1)KS1237,v/fs * os females screened (see MATERIALS AND METHODS). been more feasible. The shaking activity is not de- pendent on the state of anesthesia nor the anesthetic abnormalprofile of actionpotentials from ShKS*jj used. Flies treated with COZ,C1&, N2 or cold tem- (data not shown). Thus, it seems that the modification perature (4O C) vibrate their appendageswith the same of the shaking activity due to the B27 breakpoint characteristics as when they begin to recover from occurs downstream from the biological functions in- ether anesthesia. Also, fully awake flies manifest volved, perhaps at the neuromuscularlevel. chronic vibration as well as abnormal action poten- In summary, mutations of the ME region affect, in tials. The only differential effect detected was with different ways, oogenesis; some of them affectalso the triethylamine. The mutant ShrKO'*'seems to be hyper- differentiation of the hypoderm and/or the physiol- sensitive to this agent and treatments (see MATERIALS ogy of the CGF. The normal function(s) of MEIII AND METHODS)that cause recoverable anesthesia in possibly requires the formation of multimers. Also, other strains, are lethal to this allele. With 1 min of the viability of ME- males demonstrates that all these treatment, 60% of ShrKO'" flies die. In general, the mutations are antimorphs. strongerthe shaking activity of the allele the less The V mutants: The V region is defined by the deleterious is the anesthetic treatment with triethyla- 392 Ferriis et al. omous for each appendage. A further proof of the autonomy of the shaking phenotype is observed when an appendage is severed from a mutant fly. Under this drasticcondition, the severedleg continues to vibrate for several minutes decaying slowly. It should be noted that the severed leg contains no motorneu- ron somata, only nerve terminals and muscles, thus it can be said that the shakingactivity is autonomous to the axon-muscle structures. The HL mutants: The HL region is defined by the W32 and V7 breakpoints.A total of 16 mutations (Figure 6) have been mapped to this region. Allelo- morphism indicates the existence of at least nine com- plementation groups. Since the dominant lethal mu- tations do not allow any allelomorphism test, the actual number of complementation groups might be FIGURE9.-Allele specificity of abnormal action potentials from viable mutants of SRC. Note the two types of abnormal profiles (left even higher. There arereasons (see below) to believe us. right columns). that all functions coded in the HL region are func- tionally related. Therefore, we will describe the biol- mine. Other cases of lethal effects with this agent are ogy of HL mutants as a single group. described in TANSEYet al. (1987). As with the MEIIImutant 387, we noticedthat There is a clear allele specificity in the appendage 174/+ and 1614/+ adults have a visible shaking activ- vibration and theaction potential abnormality (Figure ity. The corresponding CGF action potentials show 9). We attempted to map by recombination viable Sh also an abnormal profile (Figure 8), suggesting that alleles with similar or different action potential char- these functions are similar to those coded in the V acteristics. A pair of alleles (ShKS'33and ShE6') with region. For the gynandromorph analysis we used the different phenotypes showed to be separable by re- mutations 1614, 174, and2288 3014 (Table 3). All of combination (Figure 6). By contrast, thecase ofShKS'33 them are visible (about 50% versus control) in these and Sh'" with similar phenotypes yielded no recom- mosaics although 174 yield very few cases(1 0% versus binants among8544 male offspring. However the case control) and with small disperse patches of mutant of ShKS13' and Sh5 with clearly different phenotypes territory. Among the mosaics obtained, abnormal po- showed norecombinants among 101 12 male off- sition of appendages and movements that could pos- spring. The existence of recombination is a definitive sibly be related to neuro/muscular defects were de- proof of a relatively distant location of the mutations. tected in 1614 and 3014 cases. The limited collection However, the lack of recombination can be due to of gynandromorphs obtained allowed us to calculate either the close proximity of the alleles in question or tentativefate maps of the lethality foci (datanot the existence of a subtle rearrangement in one of shown), In allcases (including the ME mutations), them causing defects in chromosome pairing. In the these are located in the ventral side of the embryo, latter case, the Southern blot analysis should serve to roughly the anlage where the CNS and muscles orig- detect such rearrangements. Since no DNA restriction inate (POULSON1950). Also, the viable mutation held pattern abnormalities have been detected in ShKs'33 up (hdp) has beenlocated within theHL region and Sh' flies (data notshown), it is suggested that these (HOMYK andEMERSON, 1988). We have found it to be might be closely linked point mutations. allelic to and with respect to the muscle The shaking phenotype is autonomous in mosaics. 1614 8384 phenotype.However, abnormal wing position is a We generated gynandromorphsby crossing 0 ShKS133/ common feature of aged individuals of the genotypes: FM6;candleand X 6 y w. A total of 70 experimental mosaics were obtained. The individual inspection of 0 1614/+,6 Sh', 8 ShlZoA,d V7 and d B55. It is not these mosaics showed that the shaking of any given known if these defects are of muscle or neural origin. appendage is independent from that of its contralat- For germ line mosaics we used the mutations 174, eral homolog and is associated with the mutant con- 1614,484, 3014and 2288 (Table 4). With the excep- dition of the corresponding hypodermal landmarks. tion of 3014, all the mutations tested seem to alter It should be noted that in homozygous Sh mutants the oogenesis. Two of them (174 and 2288) causing le- intense vibration of appendages shows a bilateral co- thality of the oogonia and anothertwo (161 4 and 484) ordination.However, in these mosaics and certain causing the same phenomenon as the MEIII mutant states of recovery from anesthesia in the homozygous 1929, namely the homozygous ovarioles give rise to flies, it can be seen that the shaking activity is auton- mutant survivors (Table 4). Heretoo, these males Genetic Analysis Genetic of Shaker 393 died within a few days after eclosion showing a pro- TABLE 5 gressive decay of their motor activity. Shaking activity of mutants and aneuploids The HL region was tested for its dominant haplo lethality in somatic mosaicsin Df(I)JC153/FM6; Genotvpe Sh activity Dp(1;3)JC153,Ki Sb/+ larvae irradiated72-96 h after ShK.Sl33 3 Sh’ 3 egg laying. A total of 4 notum and 13 abdomen Ki+ Shlll2 Sb+ clones were obtained indicating that the HL-/+ 3 ShM 2 condition is tolerated in hypodermal clones. Also, the ShE62 3 irradiation ofW32D/V7p; Dp(1;3)JC153 Ki Sb/+ larvae ShrKOlZO 2 yielded 2 clones in the notum (maximum size 5 bris- B55 3 tles) and 11 clones in the abdomen (maximum size 4 W32 2 bristles), in all cases without detectable abnormalities. v7 1 FM7alW32’ 1 These results indicate that the HL- condition is also FM7alB55’ 1 cell viable inthe hypoderm. The contrast between the FM7a/W32I2/B55” 1 abnormalmorphology of mutanthypoderm in the B55/W32” 3 gynandromorphs of 274,2288and 3014 and the lack V7”/W32” 2 of defects in HL- hypodermal spots, indicates that V7”/B55”/V29’ 1 V7”/B55” 2 this trait of the mutant phenotypeis not equivalent to ~32~)l85s’ 2 the corresponding deletion. In addition, it points to- B55”/W3Y 3 wards the antimorphic nature of these mutations. B55/V2Yi‘ 3 In germ line mosaics, the condition ME- HL-/ME- V2P/W32J’ 1 ShK.$l3J/W32P 3 HL+ (genotype: Of(l)JC153/Df(l)JC153/W32P)is FM7alV7” 2 lethal (0 clones in 152 females screened). Also, B55fFM7a 2 the condition ME- HL-/ME+ HL’ (genotype: B55”/W3T/FM76 3 Df(l)JC153/T(X;3)JC153)is lethal (0 clones in 512 W?Y/FM6/B75‘ 1 females screened). Theseexperiments indicate that W3ZD/B75‘ 2 B75/W3y 1 the HLregion encodes dominantlack-of-function (i.e., XXIW32‘ 1 equivalent to their deletion) properties required dur- B75”/W3Zp/FM7b 2 ing oogenesis. That is, a dominant female sterilemu- B551V.29” 2 tation would be expected in the mutant saturation of B55/V2P/W3ZP 2 this region. Possibly, the dominant lethal mutations Df (l)JCl53/FM7~/B55~ 2 Df(l)jClS?/BSS‘ 1 5051, 9916 or 13193 would fulfill this expectation, Df(l)JCl53/FM7c/W32‘ 2 albeit their dominant lethality prevents the testing of Df(I)JC153/W32‘ 2 this possibility. B55D/FM6/Dp(l; 3)jC153 2 The HL region also includes the viable re- B55”/BlSP 2 arrangement T(X;Y)V7 which shows shaking and ab- The intensityof shaking activity is rated 1 to 3 in increasing normal CGF action potential phenotypes (Figure order. Note that ME-V- (Df(I)JC153/W3y)males have a weaker 4). shaking phenotype than any of the V mutants suggesting that the The aneuploid 0 V7D/+ is viable although with a shaking activity phenotype is not equivalent to the deficiency. Also Minute phenotype due to thehaploinsufficiency of the note that the 855” element causes a dominant shaking phenotype by itself indicating that functions related to the membrane repolar- M(l)n+locus. Also, we questioned to which element ization areencoded distal to 855 andperturbed by this re- (distal or proximal) is the dominant shaking phenotype arrangement. associated?. Table 5 shows thedegree of shaking activity of various aneuploids (see also Figure 10). It that many double lethal heterozygous female combi- seems that the dominant shaking phenotypeis associ- nations give rise to male escapers in anoticeable ated with the distal as well as the proximal elements frequency. The escapers included representatives of of B55 and W32. In V7,only the distal element causes both lethals whenever the flanking markers allowed dominant shaking. their identification. These escapers lived for a short Phenotypes of trans-heterozygotes: Since the elec- time (2 days maximum) with obvious movement im- trophysiological analysis of most of the ME and HL pairments, including failure to mate normal females. lethals is still pending, we searched for more genetic Not all trans-heterozygotes give rise to viable escapers. evidence for functional relationships among the iso- The heterozygotes that yield escapers most frequently lated mutations. For that purpose we constructed all are HL/ME combinations. This effect is comparable possible pairwise combinations of lethals in heterozy- to the result obtained in the germ line clones (see gous females which were then crossed to FM6 males. above) and leads tothe same interpretation.It is In each cross we screened forthe appearance of plausible that the HL and ME products coparticipate escaper males of each lethal involved. Table 6 shows in theirnormal functions by means of multimeric 394 Fer& et al. - structures.However, in this case theformation of + ME I-L 1 hetero- rather than homomultimersis suggested DPJC63 8u) 866 DISCUSSION W32 v7 The genetic analysis of the Shaker region indicates VZS that mutations along 350 kb show either similarities FIGURE10.-Diagram of most frequently used rearrangements in their neural phenotypes, antimorphic behavior in in the analysis of ShC. gene dosage tests or maternal rescue of lethality in the accumulation of intermediaries has a toxic effect trans-heterozygotes of certain combinations.Based on while the abolition of the entire pathway is less dele- these features we refer to theinterval between JC153 terious due toalternative routes. distal and S4010 as the Shaker gene complex. Within the context of this analysis, the meaning of A previous study of the V alleles in gene dosage multimer is somewhat different from the commonuse experiments indicated that these mutations are anti- in biochemistry. We do not mean to imply a structure morphs (TANOUYE,FERR~S and FUJITA 198 1). The whose components are physically andpermanently present report on the characteristicsof the ME region bound,rather we referto the functionalcoupling and the existence of lethals in this interval also indi- between proteins that mightassemble transiently. The cates that the ME lethals are antimorphs. A further required flexibility of biological systems yet maintain- indication of antimorphism is obtained with the germ ing an exquisite coordination, possibly relies in this line clones of the ME lethal 1929 and the HL lethal kind of interaction. With the exception of some bio- 1614. The electrophysiological phenotype of the V chemical pathways, there are not many examples of alleles, the tested ME and HL lethals andthe V7 clusters of functionally related genes in eukaryotes. breakpoint is another common trait. We feel that this dearth of antecedents is only a Any proposition about the functional organization consequence of the limitations for sophisticated ge- of the ShC must account for the antimorphicbehavior netic analysis in most organisms.Nonetheless, the of all the mutations analyzed. It should be realized structural elements of muscles have received consid- that the gene dosage tests, in which the definition of erable attention.Several muscle component genesare antimorphs is based, do not provide information di- clustered in Drosophila (KARLIKet al. 1984), mouse rectly about the molecular consequences of the mu- tationalevents. The molecular basis of antimorphs (WEYDERTet al. 1985) and Caenorhabditis (LANDEL can be either modifications of a gene product so that et al. 1984). Interestingly, mutations in this latter case the novel entity perturbs the normal function, or the belong to the antimorph class. In Drosophila, myofi- abolition of the mutated gene product. In this latter bril assembly is dependent on the proper stoichiome- case, the absence of a product can behave as an try of actin and myosin molecules. Trans-heterozy- antimorph if the normal function is accomplished by gotes for the structuralgenes for these molecules show a multimer in which the product in question used to near normal muscle structure while heterozygotes of be included.We are pointingout that defects in any of them separatedly have quite abnormalmuscles product balance could, under some circumstances, (BEALL,SEPANSKI and FYRBERC1989). This observa- meet the usual criteria for antimorphism. tionmight be equivalent toours on the maternal We propose that both classes of antimorphs exist rescue of ME/HLlethal combinations. Evidences among theShC mutants. The first class refers to those from the genetic analysis of nonclusteredgenes in mutations that yield a modified gene product which, Drosophila whose mutations affect muscle biology by itself or as part of a multimer, give rise to a new seem to indicate that they are functionallyrelated biobgicalfunction. To this class belong the viable (HOMYK andEMERSON 1988; DE LA POMPA, GARCIA alleles and most EMS and ENU induced lethal muta- and FERRUS1989). The structural genes for enzymes tions in particular 1929 and I614 which yield viable involved in certain pathways are frequently clustered. lethals in germ line clones. The second class consists One such example in Drosophila is the cluster of 18 of those mutations which, we believe, yield reduced genes related to catecholamine metabolism (PENTZ, product and which are antimorphs because they alter BLACKand WRIGHT1986). No mechanistic interpre- the stoichiometry with respect to other components tation can be offered still for any of these examples. of the multimer. However, it is likely that, if the genetic analysis be- The paradox between the lethal condition of ME comes more elaborate and the studies more system- mutants and the viability of ME- flies can be under- atic, general trends on the functional organization of stood if the variousfunctions coded in this region clustered and disperse gene families will emerge. participate in a common pathway irrespective of the Our working hypothesis on the functional organi- mechanism by which this is done (e.g., multimers and zation of the Shaker complex proposes that different feed back regulation). In certain metabolic pathways, transcriptional products from ShC give rise to combi- Genetic Analysis of Shaker 395 TABLE 6 Frequencies of escapers from trans-heterozygouscombinations of Sh mutants

305 2215 1359 387 1929 459 1929 387 1359 2215 305 ShM ShKSl3’ 16142288 484 174 3014 4- 305 2215 I359 38 7 1929 45Y 0.2 ShM ShK.\lf3 2288 1 1 0.2 1614 0.4 0.20.2 0.4 7.4 484 8.615.4 7.8 8.8 1 1.2 0.23 0.4 1.2 30140.4 0.4 0.4 0.4 0.2 0.4 1.4 6.6 174 1.2 0.2 + 7 100

Numbers indicate the percentage of escapers among a minimum of 2000 offspring from crosses ? *1/*2 X 6 FM6. These crosses were used also for the measurement of recombination. Recombinants were distinguished from escapers by the exchange of flanking markers and subsequent behavior of recoveredmales. Blank spaces indicate 0 escapers except forV/V combinations which were not measured. nations of multimeric, possibly multifunctional, aggre- that aprotein kinase is coded in this region (our gates. In this context, seemingly different processes unpublished results). Even though it is not yet known such as membranerepolarization, muscle structure if this kinase actually uses the K+ channels coded in and oogenesis could have a common genetic base. the V region as a substrate, the V products contain The normal MEI, I1 and 111 products coded in the suitable sequences to be targets of phosphorylation ME region seem to be relevant during oogenesis be- (PONCS et al. 1988). cause the entire region, which contains lethal muta- The normal products codedin the V region are the tions in the three complementation groups, can be best known to date at themolecular level (SCHWARTZ obtained as a viable deficiency only when the maternal et al. 1988; PONCSet al. 1988; KAMB, TSENC-CRANK oogenesis was performed by euploid cells. Indeed, the and TANOUYE1988). It is proven that they act as K+ Northern analysis of this region has shown the abun- channels of various kinds (IVERSONet al. 1988; TIMPE dant expression of ME transcripts in oocytes (BAU- et al. 1988). The Western blots stained with antibodies MANN et al. 1987). However, it is likely that the ME against synthetic peptides show that ShM and ShKS”’ functions are not limited to oogenesis since the lethal alleles yield protein(s) with the normal M,. (BARBASet phases of different mutants occur at various stages al. 1989) supporting the interpretation of the V mu- during development and mutant phenotypes can be tations as antimorphs due to modified gene products observed in other tissues. Also, not all ME functions (TANOUYE,FERR~S andFUJITA 1981). The in situ are equivalent. For instance, the ME I+ function is hybridization of exon specific riboprobes demonstrate required for cell viability while ME ZZ+ and ZZZ+ are that all V products are expressed in the nervous not. Thelethality of allgenotypes observedafter germ system andthat most alleles do notperturb this line clones with the ME ZZ mutant 1359, suggests that expression (PONCSet al. 1988; our unpublished data). the ME ZZ+ function(s) is essential during early embry- Finally, thetransformation experiments with trun- ogenesis and not rescuable by the zygotic genome. cated cDNAs from the V region show that a normal However, the pupal lethal phase of the mutant sug- fly can be transformed into a mutant (GISSELMANet gests further, that the ME ZZ+ function(s) is required al. 1989). also at this later time of postembryonic development. Concerningthe HLregion, it is remarkablethe Interestingly,both stages have in commonmajor high number of complementing lethals found in this changes of the neural pattern. relatively shortinterval (Figures 3 and 6). The ex- The dominant enhancingeffect of B27 upon ShKS133 pected dominant lethal mutationshave been obtained and the dominant shaking phenotypes of the ME ZZZ only after X-ray mutagenesis resulting in chromo- lethal 459 and the B55Delement, suggest a functional somal rearrangements. The categories of mutations relationship between ME and V products. Possibly, in the HL interval range from viables to dominant some of the ME functions, in addition to theiressential lethals including semilethals, recessive lethals and sem- contributions to oogenesis, participate in some pro- idominant lethals. The analysis of complementation cesses where K+ currents are involved. Recent data (Fig. 6) indicates that a viable (hdp),a recessive lethal from the molecular analysis of ME products indicate (1614) and a semidominant lethal (8?84) belong to 396 Ferrk et al.

the same complementation group. We take these ob- It is a fact that K+ currents are the most diverse servations as an indication that theconstituents of the type of ionic currents (RUDY1988; HILLE1984). It is HL region might be functionally very similar (hence also a fact that certain K+ currents are key processes the common mutantphenotypes) and their functional in the modulation of synaptic efficacy (CROW1988; genomic organization be subject to quantitatively dif- BYRNE1987; WALTERSand BYRNE1983; KANDEL ferent degrees of perturbation (hence the range of and SCHWARTZ1982). To accomplish this role,a viabilities). The largenumber of complementation number of biochemical activities such as phosphoryl- units in HL might be viewed as an indication of a ation,dephosphorylation, nucleotide binding and wealth of transcription units, however, the ongoing translocation of cytoskeletal proteins must be tightly studies at the molecular level show less transcription coupled with the K+ channels (LOGOTHETISet al. units than complementation groups (our unpublished 1987; NORTHet al. 1987; ASHCROFT1988; LEVITAN data).In fact, the discrepancies between functional 1988).Furthermore it is likely that this functional complementation and physical location of 174 and couplingrequires also astructural coupling in the 1614 suggests the existence of functional links be- form of amultimer. We hypothesize thatthe ShC tween these HL components. The observation that encodes a large family of products which form multi- DL mutations have been obtained only among X-ray functional combinations in hetero- as well as homo- treated chromosomes and not among those treated multimers. Concerning the biology of K+ currents, we with EMS, suggests that a perturbationof the physical proposethat the functionalcoordination required organization of the DNA is required to yield such a among K+ channels andtheir modulatingfactors phenotype. At present, we entertain two alternative might be a consequence of the genetic organization working hypotheses on the functional organization of of the ShC (see also FERR~,LLAMAZARES and GAUN- the HL region: a) the HL+function is represented by IT2 1988). a regulatory, nontranslatable (hence not altered by There is no reason to suspect that all modulating point mutations) stretchof DNA, b)the haplolethality elements of K+ channels will be clustered within ShC. is the result of the combined depletion of a number We have isolated the gene tetanic (tta) as a possible of functionally related products. This combined de- trans-regulator of ShC expression using a gene dosage pletion would be attained only after a polar effect sensitivity test (ORGADet al. 1989). Perhaps not as a from chromosomal rearrangements. If this latter hy- surprise, tta mutations alter the levelsof a specific pothesis were correct, the large number of comple- protein phosphatase (ORGADet al. 1989) which is a mentation units could be due toprotein complemen- required modulating elementfor phosphorylated sub- tation (ZABIN and VILLAREJO1975). Another haplo- strata. lethal region of Drosophila’s genome is located in 83 The work with Shaker in Drosophila has facilitated DE, albeit in this case it is also triplolethal. A muta- the isolation of homologous K+ channels in verte- tional analysis of this region searching for dominant brates (STUHMERet al. 1988; BAUMANNet al. 1988; lethals has yielded alsomostly chromosomalre- TEMPEL,JAN and JAN 1988). Aside from the utilitarian arrangements rather than point mutations (ROEHR- aspect of this work, we feel that the studyof this gene complex and its regulation will be informative about DANZ and LuccH~sr1980). It is plausible that both cases (83 DE and16 F) sharea common genetic the functional organization of the genome as the unit with true biological significance. The genetic analysis organization. performed so far provides information about the ex- Aside from the K+ current, the precise nature of istence of relationships among theidentified elements. theother functionsencoded in ShC is still under However, we are proposingthat a higher level of investigation. The available evidence from the ongo- genomic organization exists whereby more elaborated ing studies indicate that a protein kinase is encoded features of the biology of K+ channels are specified. in the ME region and that a Ca2+binding protein and Up to now we have identified the partners maintaining a calmodulin-like protein are encoded in the HL re- functional relationships. From now on we have to gion (J. A. BARBASet al. and I. KRAH-JENTGENSet al., unravel the language that define this higher level of submittedfor publication). Furthermore, all these genetic organization. productsare expressed in the nervous system. Al- though these observations can not be taken as a dem- This report is the account of ten years of work on this subject. onstration of the hypothesis derived from the genetic It began at the California Institute of Technology, continued under analysis they are in strict agreement with it. At pres- very difficult conditions at the Centro de Biologia Molecular and ent, we can only say that most, if not all, ShC functions finished at the Instituto Cajal. We wish to thank those colleagues relate to thebiology ofK+ channels since the abnormal who helped and encouragedus during these years in various ways, most notably: S. BENZERand J. MODOLELL.Also we are in debted CGF action potentials found in several mutants (387, to J. MERRIAMand G. LEFEVREwho provided essential chromo- 1614, 174, V7) are similar to those exhibited by the somal rearrangements. Our colleagues in the laboratories of Madrid V mutations. and Bochum contributed with their criticisms. Funds were obtained Genetic Analysis of Shaker 397

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